LOCUS       sequence-361472-        6923 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gives very high editing efficiency in barley. Also works in Brassica
            oleracea.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6923)
  AUTHORS   Lawrenson T, Hinchliffe A, Forner M, Harwood W
  TITLE     Highly efficient genome editing in barley using novel LbCas12a 
            variants and impact of sgRNA architecture
  JOURNAL   bioRxiv 2022.04.28.489853
REFERENCE   2  (bases 1 to 6923)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            2022.04.28.489853"
COMMENT     Sequence Label: ttAtCas12a+int
FEATURES             Location/Qualifiers
     source          1..6923
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(36..54)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        122..226
                     /gene="bla"
                     /label=AmpR promoter
     CDS             448..1239
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     rep_origin      1410..1998
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1899..1918
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2152..2169
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             6869..6916
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
ORIGIN
        1 tgagaccacg aagtggctct tcagtggacg aaagggcctc gtgatacgcc tatttttata
       61 ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt
      121 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
      181 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatgc gctcacgcaa
      241 ctggtccaga accttgaccg aacgcagcgg tggtaacggc gcagtggcgg ttttcatggc
      301 ttgttatgac tgtttttttt tggggtacag tctatgcctc gggcatccaa gcagcaagcg
      361 cgttacgccg tgggtcgatg tttgatgtta tggagcagca acgatgttac gcagcagggc
      421 agtcgcccta aaacaaagtt aaacatcatg agggaagcgg tgatcgccga agtatcgact
      481 caactatcag aggtagttgg cgtcatcgag cgccatctcg aaccgacgtt gctggccgta
      541 catttgtacg gctccgcagt ggatggcggc ctgaagccac acagcgatat tgatttgctg
      601 gttacggtga ccgtaaggct tgatgaaaca acgcggcgag ctttgatcaa cgaccttttg
      661 gaaacttcgg cttcccctgg agagagcgag attctccgcg ctgtagaagt caccattgtt
      721 gtgcacgacg acatcattcc gtggcgttat ccagctaagc gcgaactgca atttggagaa
      781 tggcagcgca atgacattct tgcaggtatc ttcgagccag ccacgatcga cattgatctg
      841 gctatcttgc tgacaaaagc aagagaacat agcgttgcct tggtaggtcc agcggcggag
      901 gaactctttg atccggttcc tgaacaggat ctatttgagg cgctaaatga aaccttaacg
      961 ctatggaact cgccgcccga ctgggctggc gatgagcgaa atgtagtgct tacgttgtcc
     1021 cgcatttggt acagcgcagt aaccggcaaa atcgcgccga aggatgtcgc tgccgactgg
     1081 gcaatggagc gcctgccggc ccagtatcag cccgtcatac ttgaagctag acaggcttat
     1141 cttggacaag aagaagatcg cttggcctcg cgcgcagatc agttggaaga atttgtccat
     1201 tacgtaaaag gcgagatcac caaggtagtc ggcaaataac tgtcagacca agtttactca
     1261 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     1321 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     1381 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     1441 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     1501 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     1561 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     1621 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     1681 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     1741 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     1801 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     1861 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     1921 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     1981 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     2041 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     2101 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     2161 gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg
     2221 attcattaat cactctgtgg tctcaaatga gcaagctcga gaagtttacc aactgctaca
     2281 gcctctctaa gaccctcagg ttcaaggcta tccctgtggg aaagacccaa gagaatatcg
     2341 acaacaagag gctcctcgtc gaggatgaga agagagctga agattacaag ggcgtgaaga
     2401 agctcctcga caggtactac ctcagcttca tcaacgatgt gctccacagc atcaagctca
     2461 agaacctcaa caactacatc agcctcttcc gtaagaaaac caggaccgag aaagagaaca
     2521 aagagcttga gaacctcgag atcaacctcc gtaaagagat cgccaaggct ttcaagggaa
     2581 acgagggata caagagcctc ttcaagaagg atattatcga gacaatcctg cctgagttcc
     2641 tggacgataa ggatgagatc gctctcgtga acagcttcaa cggattcact actgccttca
     2701 ccggattctt cagaaacagg gaaaacatgt tcagcgaaga ggccaagagc acctctatcg
     2761 ctttcagatg catcaacgag aacctcacgc gttacatcag caacatggac atcttcgaga
     2821 aggtaacatt ccttagttac ctttcttttc tttttccatc ataagtttat agattgtaca
     2881 tgctttgaga tttttctttg caaacaatct caggtggacg ccatcttcga taagcacgag
     2941 gtgcaagaaa tcaaagagaa gatcctcaac agcgactacg acgtcgagga cttttttgaa
     3001 ggggagttct tcaacttcgt tctcacccaa gagggcatcg acgtgtacaa cgctattatc
     3061 ggaggattcg tgaccgagtc tggggagaag attaagggac tcaacgagta catcaacctg
     3121 tacaaccaga aaacgaagca gaagctcccg aagttcaagc cgctctacaa gcaggtctgt
     3181 ctttcctatt tcatatgttt aatcctagga atttgatcaa ttgattgtat gtatgtcgat
     3241 cccaagactt tcttgttcac ttatatctta actctctctt tgctgtttct tgcaggttct
     3301 ctctgatcgt gagagcctct cattttacgg tgagggttac acctctgacg aggaagtgct
     3361 tgaggttttc cgtaacaccc tcaacaagaa cagcgagatc ttctcgtcca tcaagaagtt
     3421 ggagaagctt ttcaagaact tcgacgagta cagcagcgct gggatcttcg ttaagaacgg
     3481 acctgctatc agcaccatca gcaaggatat tttcggcgag tggaacgtga tcagggacaa
     3541 gtggaatgct gagtacgatg acatccacct caagaagaag gctgtcgtca ctgagaagta
     3601 cgaggatgac aggcgtaagt cgttcaagaa gatcggctct ttcagcctcg agcagcttca
     3661 agaatacgct gatgctgatc tcagcgtggt cgagaagctc aaagagatca tcatccagaa
     3721 ggtcgacgag atctacaagg taagttgtta cttatgattg ttttcctctc tgctacatgt
     3781 attttgttgt tcatttctgt aagatataag aattgagttt tcctctgatg atattattag
     3841 gtgtacgggt cctctgagaa gttgttcgat gctgatttcg tcctcgagaa gagtctgaag
     3901 aagaacgacg ctgtcgtcgc gatcatgaag gatttgctcg acagcgtgaa gtccttcgag
     3961 aactatatca aggccttctt cggagagggc aaagagacta atagggacga gtctttctac
     4021 ggggatttcg tgctcgctta cgatatcctc ctcaaggtgg accatatcta cgacgccatc
     4081 agaaactacg tgacccagaa gccttacagc aaggacaagt tcaagttgta ctttcagaac
     4141 ccgcagttca tgggcggatg ggacaaagac aaagagacag attacagggc caccatcctc
     4201 aggttagtat catatgaaga aatacctagt ttcagttgat gaatgctatt ttctgacctc
     4261 agttgttctc ttttgagaat tatttctttt ctaatttgcc tgatttttct attaattcat
     4321 taggtacggg tctaagtact acctggccat catggacaag aaatacgcca agtgcctcca
     4381 aaagatcgac aaggatgacg tgaacgggaa ctatgagaag atcaactaca agctccttcc
     4441 gggaccgaac aagatgcttc ctaaggtgtt cttcagcaag aaatggatgg cctactacaa
     4501 cccgtctgag gacatccaga aaatctacaa gaacgggacc ttcaagaaag gcgacatgtt
     4561 caacctcaac gactgccaca agctcatcga tttcttcaag gacagcatct cgcgttaccc
     4621 gaagtggtct aacgcttacg actttaactt cagcgagaca gaaaagtaca aggatatcgc
     4681 cgggttctac cgtgaggttg aggaacaggg ttacaaagtt agcttcgaga gcgcctccaa
     4741 gaaagaggta aatcctggtc cacactttta cgataaaaac acaagatttt aaactatgaa
     4801 ctgatcaata atcattccta aaagaccaca cttttgtttt gtttctaaag taatttttac
     4861 tgttataaca ggttgacaag ttggtcgaag agggcaagct ctacatgttc cagatctata
     4921 acaaggactt ctccgacaag agccacggaa ctcctaacct ccatacgatg tacttcaagc
     4981 tgcttttcga cgagaacaac cacgggcaga tcagactttc tggtggtgct gaactcttca
     5041 tgcgtagggc ctcactcaag aaagaagagt tggttgttca cccggccaac tctccaatcg
     5101 ctaacaagaa tcctgacaac ccgaaaaaga ccaccacgct gtcttacgac gtctacaagg
     5161 acaaaaggtt cagcgaggac cagtacgagc ttcatatccc gatcgctatc aacaagtgcc
     5221 cgaagaacat cttcaagatc aataccgagg taaggacttc tcatgaatat tagtggcaga
     5281 ttagtgttgt taaagtcttt ggttagataa tcgatgcctc ctaattgtcc atgttttact
     5341 ggttttctac aattaaaggt gagggtgctg ctcaagcacg atgataaccc ttacgtgatc
     5401 ggaatcgatc gtggtgagag aaacctcctc tacatcgttg tggtggacgg aaagggaaac
     5461 atcgtcgagc agtacagcct gaacgagatt atcaacaatt tcaacggcat caggatcaag
     5521 accgactacc actcactcct cgataagaaa gaaaaagagc gtttcgaggc caggcagaac
     5581 tggacttcta tcgaaaacat caaagagttg aaggccggct acatctctca ggtggtgcat
     5641 aagatctgcg agctggtgga aaagtacgat gctgtgatcg ctcttgagga cctcaactct
     5701 gggttcaaga acagtagagt gaaggtaagt tctgcatttg gttatgctcc ttgcatttta
     5761 ggtgttcgtc gcacttccat ttccatgaat agctaagatt ttttttctct gcattcattc
     5821 ttcttgcctc agttctaact gtttgtggta tttttgtttt aattattgct acaggttgag
     5881 aagcaggtct accaaaagtt cgagaagatg ctcatcgaca agctcaacta catggtggac
     5941 aaaaagagca acccttgcgc taccggtggt gctcttaagg gataccagat cacgaacaag
     6001 ttcgagtcct tcaagagcat gagcacccag aacggcttca tcttctatat ccctgcttgg
     6061 ctcaccagca agatcgatcc ttctactggt ttcgtgaacc tgctcaagac caagtacacc
     6121 tcgatcgccg acagcaagaa gttcatctcg tctttcgaca ggatcatgta cgtgccggaa
     6181 gaggatcttt tcgagttcgc tctcgactat aagaacttca gcaggaccga cgccgactac
     6241 attaagaagt ggaagctcta ctcctacggg aaccgtatca ggatcttccg aaatccgaag
     6301 aaaaacaacg tgttcgactg ggaagaagtg tgcctcacct ctgcctacaa agaactgttc
     6361 aacaagtacg gcatcaacta ccagcagggt gatatcaggg ctcttttgtg cgagcagagc
     6421 gacaaggcat tctacagctc attcatggcc ctcatgtctc tcatgctcca gatgaggaac
     6481 tctatcaccg gaaggaccga tgtggacttc cttatctctc cggtcaagaa ctctgacggg
     6541 atcttctacg acagccgtaa ctatgaggct caagagaacg ctatcctgcc gaagaatgct
     6601 gatgcaaacg gggcttacaa cattgcgaga aaggtaaagc aactgtgttt taatcaattt
     6661 cttgtcagga tatatggatt ataacttaat ttttgagaaa tctgtagtat ttggcgtgaa
     6721 atgagtttgc tttttggttt ctcccgtgtt ataggttctc tgggctatcg ggcagtttaa
     6781 gaaagcggaa gatgagaagc tggacaaggt gaagatcgcc atctccaaca aagagtggct
     6841 tgagtacgct cagacctccg ttaagcacaa gaggcctgct gctactaaga aagctggtca
     6901 ggctaagaag aagaaatgag ctt
//
